The metabolites of the sex and stress hormones pregnenolone, progesterone, desoxycortico-sterone, cortisone and cortisol, known as pregnanolones; as well as the metabolites of testosterone, androstendione and dehydroepiandrosterone, have all been the subject of various studies, at least partially elucidating their role in the neurological signal system in mammals.
The steroids inducing CNS symptoms and disorders of interest in the present application all have a structural similarity in comprising a 3alpha-hydroxy group, a 5alpha or 5beta pregnane steroid body, and a ketone or hydroxy group on position 17, 20 or 21.
Steroids comprising 3alpha-hydroxy-5alpha/beta-pregnan-20-one/ol or 3alpha-hydroxy-5alpha/beta-androstan-17-one/ol have been shown to be important specific enhancers of the gamma-aminobutyric acid (A) receptor (GABA-A). They bind to the GABA-A receptor and act by enhancing the effect of GABA in terms of the opening frequency of the GABA-A receptor and its opening duration. The effect is similar to the effects of both benzodiazepines and barbiturates. Said steroid compounds however have a binding site separate from that of both these compounds. Examples of such GABA enhancing steroids and their number according to the Chemical Abstracts Registry/Chicago Academy of Science (CAS) are given in Table 1.
The steroid nomenclature is not entirely consistent, and therefore the nomenclature developed by the International Union of Pure and Applied Chemistry (IUPAC) will be used throughout this application.
TABLE 1Nomenclature of GABA-steroidsIUPAC - nomenclatureCAS Number3alpha-hydroxy-5alpha-pregnan-20-one516-54-13alpha-hydroxy-5beta-pregnan-20-one128-20-13alpha,21-dihydroxy-5alpha-pregnan-20-one567-02-23alpha,21-dihydroxy-5beta-pregnan-20-one567-03-33alpha,11beta,17alpha,21-tetrahydroxy-5beta-53-02-1pregnan-20-one3alpha-11beta,17alpha,21-tetrahydroxy-5alpha-302-91-0pregnan-20-one3alpha-17alpha,21-trihydroxy-5alpha-pregnan-547-77-311,20-dione3alpha-17alpha,21-trihydroxy-5beta-pregnan-53-05-411,20-dione3alpha-hydroxy-5alpha-androstan-17beta-ol1852-53-53alpha-hydroxy-5beta-androstan-17beta-ol—*3alpha-hydroxy-5alpha-androstan-17-one53-41-83alpha-hydroxy-5beta-androstan-17-one53-42-9*CAS Number not found
Some of these steroids have been shown to have an ability to induce anesthesia at a high pharmacological dose. They can also be used as anti-epileptic agents, or as soporific agents. Some of these compounds have also been shown to possess anxiolytic effects in animal experiments. To reach these effects, however, high concentrations or high doses are required. Additionally, they appear as acute effects.
With respect to their direct CNS effects, as mentioned above, these compounds are similar to benzodiazepins and barbiturates. However, they also have similar adverse effects as normally associated with benzodiazepins and barbiturates. The adverse effects of the endogenous 3alpha-hydroxy-pregnan-20-one-steroids or 3alpha-hydroxy-androstan-steroids are the basis for the negative CNS effects induced by these steroids. As the 3alpha-hydroxy-pregnane-steroids and 3alpha-hydroxy-androstan-steroids are endogenously produced and are metabolites of steroid hormones, some of them essential for life, their production cannot easily be interrupted. These steroids are produced in high amounts during several days to weeks during the luteal phase of the menstrual cycle, i.e. after the release of an ovum from a mature ovarian follicle, during pregnancy and during stress. They are also produced within the brain.
Diseases Caused by 3Alpha-Hydroxy-Steroids
Disorders that are caused by the action of endogenously produced 3alpha-hydroxy-5alpha steroids or 3alpha-hydroxy-5beta steroids on the GABA-A receptor are well characterized and understood. It is also known that 3alpha-hydroxy-5alpha/beta-steroids can induce tolerance to themselves and to other similar substances after exposure, and that withdrawal symptoms occur at withdrawal of the 3alpha-hydroxy-5alpha/beta-steroids. In summary, it is generally known that 3alpha-hydroxy-5alpha/beta-steroids cause CNS disorders through the above-described three possible mechanisms: a) direct action, b) tolerance induction, and c) withdrawal effect. These mechanisms will be discussed in closer detail below.
a) Direct Action
It is established that 3alpha-hydroxy-5alpha/beta-steroids can directly cause inhibition of CNS functions. Examples of symptoms caused by the direct action of 3alpha-hydroxy-5alpha/beta-steroids are sedation, tiredness, memory disturbance, learning disturbance, disturbance of motor function, clumsiness, increased appetite and food cravings, negative mood as tension, irritability and depression, which are the cardinal symptoms in the premenstrual dysphoric disorder, premenstrual syndrome and the worsening of Petit Mal epilepsy. Examples of this direct action can also be divided into sedative and anesthetic effects; disturbance of motor function; effects on cognitive function, memory and learning; worsening of Petit Mal epilepsy; premenstrual symptoms; mood changes; induction of anxiety in test animals; hyperphagia and increased appetite; food cravings etc.
b) Tolerance
Continuous and long exposure to 3alpha-hydroxy-5alpha/beta-steroids causes malfunctioning of the GABA-A receptor system. A tolerance develops and this tolerance is the initial step in a process that ultimately leads to stress sensitivity, concentration difficulties, and loss of impulse control and depression. The action of 3alpha-hydroxy-5alpha/beta-steroids has also been found to be a factor, which reinforces drug dependency. This has been the focus of extensive research. The following themes have hitherto been the main subject of research: down regulation and decreased GABA function after long-term secretion of high amounts of 3alpha-hydroxy-5alpha/beta steroids; reduced benzodiazepine and 3alpha-hydroxy-5beta steroid sensitivity in PMS; and dependence induction.
c) Withdrawal Symptoms
A continuous but shorter exposure to 3alpha-hydroxy-5alpha/beta-steroids results in a withdrawal effect when the exposure is ended. This phenomenon occurs for example naturally, during menstruation when the production of 3alpha-hydroxy-5alpha/beta-steroids by the corpus luteum of the ovary is interrupted. This withdrawal phenomenon also occurs after giving birth (postpartum) when the 3alpha-hydroxy-5alpha/beta-steroid production by the placenta is interrupted. The same phenomenon is also noted when a period of stress is ended, e.g. when work-related stress is interrupted during weekends. As a response to stress, the adrenals have produced 3alpha-hydroxy-5alpha/beta-steroids. When this production is interrupted, withdrawal symptoms may occur.
Examples of conditions that are influenced by this withdrawal phenomenon are partial epilepsy where the patient has an epileptic focus in the cerebral cortex where a worsening occurs at the withdrawal period during menstruation. This phenomenon is called “catamenial epilepsy”. Other examples are menstrual related migraine, stress related migraine, and mood changes post partum. Similar symptoms and conditions are induced during treatment with steroid hormones, such as used in oral contraceptives, postmenopausal hormone replacement therapy, steroid treatment for inflammatory diseases and during intake of anabolic/androgenic steroids. The mechanisms are similar in the induction of direct effects, tolerance development and withdrawal.
Disorders Caused by Gamma-Aminobutyric Acid (GABA) Overstimulation
High stimulation by GABA itself is in certain situations known to inhibit learning and memory function. An overproduction of GABA will worsen short-term learning and memory function. This is clinically relevant especially in elderly people. In such a situation, an antagonism of GABA's effect could be beneficial, and potentially useful in the treatment of memory disturbance. However, if the GABA antagonism is too strong, it can be dangerous for the patient, e.g. by causing convulsions. Therefore it is of interest to find specific antagonists that selectively block the GABA-steroid induced conditions and antagonists that also affect GABA-activated chloride flux in the GABA-A receptor, but which are inherently unable to totally block the GABA-A receptor.
The present inventors have surprisingly found substances that mainly antagonize GABA's own effect and other that mainly affect GABA-steroid action on the GABA-A receptor. It is important to distinguish between these effects, as an antagonism of GABA-steroid action is indicated in other situations than a direct partial antagonism of GABA's own effect.
Currently, the treatment of Alzheimer's disease (AD) and Mild Cognitive Impairment (MCI) is largely unrealized, with no preventive or curative therapies. The fact that GABA-A receptor agonists like GABA-steroids, benzodiazepines and alcohol are amnesic, and that the GABA-A receptors in brain areas most affected by AD, highlighted the GABA-A receptor as a potential therapeutic target in AD and MCI. In contrast to the amnesic GABA-A receptor agonists, inverse agonists or antagonists will attenuate GABA-A receptors function. GABA-Steroid antagonists and GABA antagonists have been shown to improve performance in animal models of learning and memory. Unfortunately, non-selective and totally blocking ligands also induce convulsions. Thus, there is a need for partial and selective blockers (Maubach, K., GABA-A receptor subtype selective cognition enhancers, Curr. Drug Targets CNS Neurol Disord., 2003 (2) 233-239). The current cognition enhancers marketed have prominent adverse effects with minimal efficacy and there is a need for new substances, which is the subject of the present invention.
Prior Art
To the best knowledge of the present inventors, steroid substances that selectively inhibit GABA-steroid action or mainly inhibit GABA action on the GABA-A receptor have not been disclosed earlier. The prior art only describes a general action on all GABA-steroid effects.
Prince and Simmons (Neuropharmacology, vol. 32, no. 1, pp. 59-63, 1993) have used a model relying on membrane fractions of whole male rat brain. In this sub-fraction of whole brain homogenate, the authors used the binding of a benzodiazepine, 3H-flunitrazepam, as a model for steroid effect and change of GABA-A receptor conformation. This test has been suggested as an indicator of allosteric modulation of the GABA-A receptor. The relationship between the change in flunitrazepam (FNZ) binding and change in chloride flow at GABA-stimulation is however uncertain and a change in binding can not be taken as a proof of a change in the chloride flow through the GABA-receptor, nor as a proof of a change in GABA-A receptor function. The change in chloride flow is the important effect.
The central question, i.e. if there exists a relationship between change in FNZ-binding and neuronal excitability, is even less clear and such conclusions cannot be drawn from results on FNZ-binding alone. A change in FNZ-binding properties or absence of such change in binding properties does not imply a change or the absence of a change in neural activity or GABA-A mediated chloride flow.
It is also well known that the GABA-A receptor contains several sub-units that can be combined in multiple ways. Interestingly, certain combinations lack steroid recognition site. It is also known that the effect of steroid on the binding of a convulsant substance TBPS (t-butylbicyclo-phosphorothionate) differs in different brain regions. Further, it is known that the binding of TBPS varies with the oestrus cycle in female rats indicating an effect change related to the ovarian hormone production. These changes related to the oestrus cycle can of course not be noticed in male rats, as used in the studies of Prince and Simmons (supra).
U.S. Pat. No. 5,232,917 (Bolger et al.) and U.S. Pat. No. 5,939,545 (Upasani et al.) disclose a number of 3alpha-hydroxy steroids. Both these patents concern the agonistic modulation of the GABA-A receptor. In other words, these patents are both focused on the benzodiazepine-like effect of the 3alpha-hydroxy-5alpha/beta-steroids. All steroids that are modulators of the GABA-A receptor have the common feature of one 3alpha-hydroxy structure. The 3beta-hydroxy steroids mentioned in these documents were only used as controls, in order to show that the 3alpha-hydroxy-steroids were specific. Steroids with only a 3beta-hydroxy structure have never been shown to possess a GABA-A receptor modulating effect. In all cases where an effective GABA-A receptor-modulating effect is noticed, the steroid has a 3alpha-hydroxy group.
WO 99/45931 (Bäckström & Wang) discloses the antagonistic effect of one particular steroid, namely 3beta-hydroxy-5alpha-pregnan-20-one but is silent about other 3beta-hydroxy-steroids.
The antagonistic effect of 3beta-OH-5alpha-pregnan-20-one against 3alpha-OH-5alpha/beta-pregnan-20-one was first disclosed by Wang et al. (Wang M. D., Bäckström T and Landgren S. (2000) The inhibitory effects of allopregnanolone and pregnanolone on the population spike, evoked in the rat hippocampal CA1 stratum pyramidale in vitro, can be blocked selectively by epiallopregnanolone. Acta Physiol Scand 169, 333-341). In that paper, a dose dependent antagonistic effect of 3beta-OH-5alpha-pregnan-20-one on two of the 3alpha-OH-5alpha/beta-steroids was shown.
WO 03/059357 (Bäckström et al.) discloses the use of pregnane steroids in the treatment of CNS disorders. This document does not mention the possibility to selectively inhibit GABA-steroid effects or the action on GABA itself.
That 3beta steroids also can have effects on the GABA's own effect was disclosed by Wang et al. (Mingde Wang, Yejun He, Lawrence N. Eisenman, Christopher Fields, Chun-Min Zeng, Jose Mathews, Ann Benz, Tao Fu, Erik Zorumski, Joe Henry Steinbach, Douglas F. Covey, Charles F. Zorumski, and Steven Mennerick. 3beta-Hydroxypregnane Steroids Are Pregnenolone Sulfate-Like GABAA Receptor Antagonists. The Journal of Neuroscience, May 1, 2002, 22(9):3366-3375). In this paper, the authors show that when maximal stimulation of GABA-A receptors is made with GABA, certain 3beta-hydroxypregnane steroids are able to inhibit GABA's own effect. But that certain 3beta-hydroxy steroids would mainly inhibit GABA-steroid effects on GABA gated chloride flux and other steroids would also inhibit GABA's own effect was not discovered.
It remains a challenge to find specific antagonists of the 3alpha-hydroxy-pregnan-steroid action that have low GABA inhibitory effects, and to find partial antagonists of the GABA action which compounds are physiologically safe and suitable for pharmaceutical use, and which additionally are applicable in physiologically acceptable doses.
One objective of the present invention is thus to identify such specific blockers for GABA-steroid antagonism and partial antagonism towards GABA itself and to make available novel pharmaceuticals and methods for the treatment, alleviation or prevention of steroid related and/or steroid induced CNS disorders.
Further objectives, the associated solutions and their advantages will be obvious to a skilled person upon familiarizing himself with the description, examples and claims.